Nao Suzuki, M.D, Ph.D Dept. of OB & GY, School of Medicine, St. Marianna University
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1 The 5 th World Congress of the ISFP Vienna, AUSTRIA Session 10 Storing ovarian tissue Debate- Slow freezing vs vitrification Vitrification Nao Suzuki, M.D, Ph.D Dept. of OB & GY, School of Medicine, St. Marianna University
2 COI disclosure Nao Suzuki, MD, PhD Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa, Japan In conjunction with subject announcement, there are not the companies in the COI relations that I should disclose.
3 Ovarian tissue cryopreservation Kuwayama et al: Reproductive Bio Medicine Online 2005
4 Ovarian tissue cryopreservation Vitrification Slow freezing Infiltration of CA Slow-freezing by program freezer LN 2 Transfer to cryotube Store at LN 2 tank ovarian tissue Infiltration of CA Freezing from out-side and ice crystal formation Cell damage following grow ice crystal of out-side
5 Vitrification Ovarian tissue cryopreservation Infiltration of cryoprotective agent (CA) Setting piece of ovarian tissue to vitrification device rapid-freezing by LN 2 Store at LN 2 tank Infiltration of vitrify solution (VS) LN 2 ovarian tissue Slow freezing Infiltration of CA Infiltration of CA Infiltration of VS Rapidly freezing and ice crystal formation Slow-freezing by program freezer LN 2 Transfer to cryotube Store at LN 2 tank ovarian tissue Infiltration of CA Freezing from out-side and ice crystal formation Cell damage following grow ice crystal of out-side
6 Reprod BioMed Online, 2011 Slow freezing vs vitrification
7 Slow freezing vs vitrification Reprod BioMed Online, 2011 Suitable Vitirification solutions? Toxicity Feasibility Penetration of the entire tissue Requires good training in the beginning Thinner is the best Risk in technical expertise
8 Human Reproduction 2009
9 Ovarian tissue vitrification Human Reproduction 2009 Vitrification Control 5min Slow freezing 10min Vitirification EG + DMSO + PrOH + Sucrose The ovarian stroma was significantly better preserved after vitrification than after slow freezing. On the other hand, the follicles were similarly preservaed after all freezing methods.
10 Ovarian tissue vitrification RBM Online, 2009 Vit LN 2
11 Human Reproduction 2013 VEG VEG VEG: 25% EG+ 25% Glycerol + 0.2% PVP + 0.2% PVA + 0.4% PG VED: 25% EG+ 25% DMSO + 0.2% PVP + 0.2% PVA + 0.4% PG
12 JARG, 2015 Slow freezing vs vitrification 10% DMSO+26% EG+2.5%+PV P +1M sucrose Methods of OTC Fresh ovarian tissue Slow freezing Vitrification vs Grafting sites A healed bed Freshly decorted bed vs
13 Slow freezing vs vitrification JARG, 2015 Follicle stage (%) Proliferation rate (%) vitrified tissue grafted to a freshly decorticated vascular bed vitrified tissue grafted to a freshly decorticated vascular bed
14 Slow freezing vs vitrification JARG, 2015 Follicle stage (%) Proliferation rate (%) The best combination of treatments, cryopreservation, and graft site preparation was vitrification and grafting to the freshly decorted bed. vitrified tissue grafted to a freshly decorticated vascular bed vitrified tissue grafted to a freshly decorticated vascular bed
15 Slow freezing vs vitrification Fertility and Sterility, 2016 SF: ethyl vinyl acetate bag SF vs Vit VT1: VS1 and metaric grids VT2: VS2 and metaric grids VT3: VS1 and ethyl vinyl acetate bag VT4: VS2 and ethyl vinyl acetate bag VS1: 20% EG+20% DMSO+0.5M sucrose VS2: 10% EG+10% DMSO+10% PrOH+10%PVP
16 Slow freezing vs vitrification Fertility and Sterility, 2016 Histologic evaluation and follicular population In vitro study control SF: ethyl vinyl acetate bag VT1: VS1 and metaric grids VT2: VS2 and metaric grids VT3: VS1 and ethyl vinyl acetate bag VT4: VS2 and ethyl vinyl acetate bag
17 Slow freezing vs vitrification Fertility and Sterility, 2016 Cell death Histologic evaluation and follicular population In vitro study VT1: VS1 and metaric grids VS1: 20% EG+20% DMSO+0.5M sucrose
18 Slow freezing vs vitrification Fertility and Sterility, 2016 Tissue viability Histologic evaluation and follicular population In vitro study VT2: VS2 and metalic grids The combination of a metallic grid with the VT1 solution obtained significantly better results than the other experimental groups.
19 Slow freezing vs vitrification Fertility and Sterility, 2016 PF density In vivo study fresh SF VT1 Fibotic area SF Human ovarian tissue using EG, DMSO, sucrose, and SSS does not affect the normal morphology of oocytes, follicles, and blood vessel distribution after transplantation. Ovarian tissue vitrifiation offers greater efficiency than slow freezing. VT1
20 Ovarian tissue vitrification Pre-clinical study: since M (35% v/v) EG + 5% PVP M sucrose 5min Human Reproduction 2012 Collaborative research with Dr. Shu Hashimoto and Dr. Yoshiharu Morimoto (IVF Namba Clinic, Osaka, Japan)
21 Ovarian tissue vitrification & TP: Disease Pts OTC Pts OTC Breast Cancer (BC) 64 3 SLE 4 Uterine cervical cancer (SCC) Ewing sarcoma 1 87 Leukemia 5 Malignant Lymphoma 4 MDS 3 others 4 2 Pts TP POI Total Pts.: Number of patients, OTC: ovarian tissue cryopreservation, TP: transplantation
22 Ovarian tissue vitrification: application to the new treatment for the POI patient Patient Onset of POI AMH Age when IVA was done Pregnancy outcome 23y 33y 38y 29y 31y 28y 38y 4.9 pmol ng/ml <0.01 ng/ml <0.01 ng/ml <0.16 ng/ml <0.1 ng/ml <0.01 ng/ml 25y 33y 40y 31y 36y 30y 40y Live birth Live birth Chemical abortion Abortion Abortion Live birth Abortion Three live-births were achieved using vitrified-thawed ovarian tissue. Kawamura K, Suzuki N et al: PNAS 2013 Suzuki N, et al. Human Reproduction 2015
23 Research Grant Program on the survey for children and child care promotion on the Ministry of Health, Labor and Welfare in Japan Research about the effectiveness of Oncofertility treatments for the CAYA cancer patients By Dr. Seido Takae(St. Marianna University) and Nao Suzuki (PI) The number of OTC procedures in Japan 201 in total as of
24 Research Grant Program on the survey for children and child care promotion on the Ministry of Health, Labor and Welfare in Japan Research about the effectiveness of Oncofertility treatments for the CAYA cancer patients By Dr. Seido Takae(St. Marianna University) and Nao Suzuki (PI) The number of OTC procedures according to the disease in Japan 1. Breast Cancer: Luekemia: Malignant Lymphoma: Soft tissue tumor(sarcoma): 8 5. Brain tumor: 7
25 Patients with breast cancer who underwent ovarian tissue transplantaion Breast Cancer(Luminal type): 39yo (at the time of a diagnosis) 0G0P unmarried <FH> n.p. 201X.7: Diagnosis of breast cancer ct2n1m0 cstageⅡb Luminal type 201X.9: Marriage ovayectomy and OTC, AMH: 28.4pMol/L 201X.10~: NAC(EC DTX) cpr 201X+1.3 Mastectomy + armpit lymph node dissection yct2n1m0;ycstageⅡb, Luminal type 201X+1.4~ Hormonal therapy (TAM+GnRHa:TAM: until 201X+5.2) 201X+5.9:44yo, Laproscopic ovarian tissue transplantation pre-surgery FSH: 59.8 miu/ml, E2: <25 pg/ml, AMH: <0.01ng/ml pst-surgery Menstrual cycle improvement (pre:85.0±19.7 days intervals post:41.6±17.4days intervals), AMH: 0.02ng/ml, egg collection: two times but no eggs
26 Ovarian Tissue Vitirification: New closed-type device: Cryosheet open-type closed-type 1 closed-type 2 Cooling Speed: -19,500 /min closed-type 3 New closed-type device: Cryosheet
27 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Improving the cooling speed Problem and improvement Conventional device (closed-type 2) The distance between ovarian tissue and liquid nitrogen is too great, therefore freezing is slow (<1,000 C/minute) Air LN 2 Ova tissue Improved device (closedtype 3: Cryosheet) Freezing could be speeded up by making the distance between ovarian tissue and liquid nitrogen shorter. LN 2 Ova tissue
28 Ovarian Tissue Vitirification: New closed-type device: Cryosheet The sealed pouch Cryosheet in the bag Vitrification!
29 Temperature[ ] Ovarian Tissue Vitirification: New closed-type device: Cryosheet Cooling Speed Cryo Device TypeM:-19,500 /min Closed-type 2:-13,00 /min Closed-type 3 (Cryosheet):-12,000 /min Heating Speed Cryo Device TypeM: 42,000 /min Closed-type 3 (Cryosheet):42,000 /min Time[s]
30 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Donor ovary Vitrification After 2-3 weeks orthotopic implantation (2 piece / mouse) Green mouse C57BL/6J -Tg(CAG-GFP) Half cut Wild type mouse Control (Fresh implantation) C57BL/6J In Vivo fertility assay
31 Average of days Bearing / Mating Average of live birth Ovarian Tissue Vitirification: New closed-type device: Cryosheet Results : In Vivo fertility assay The mean days until estrous cycle recovery 10 ns (%) 80 Birthrate 5/6 5 Live birth ns / control (n=7) vitrification (n=10) 0 control vitrification 0 control (n=5) vitrification (n=6)
32 Ovarian Tissue Vitirification: New closed-type device: Cryosheet cynomolgus monkeys: n=6 2 Omentum 3 Ovarian Tissue 4 1 uterus Area 1,4: Mesosalpinx 2,3: Omentum Type M-II Device 500μm Titanium device 800μm Titanium device Size and Shape Small Pieces 1,2: Right side: Sheet 2,3: Left side:
33 PrototypeⅡ (Titanium) 500μm device No.17 Ovarian Tissue Vitirification: New closed-type device: Cryosheet 30days Hyperstimulation IVF Progesteron Estradiol 500μm device No.14 Estradiol 60days Hyperstimulation IVF Progesteron Cryo-Thawing Inplantation Cryo-Thawing Inplantation 500μm device No days Hyperstimulation IVF Estradiol Progesteron μm device No.18 Hyperstimulation IVF 140days Progesteron Estradiol Cryo-Thawing Inplantation Cryo-Thawing Inplantation
34 Ovarian Tissue Vitirification: New closed-type device: Cryosheet Retrieved Oocytes Mesosalpinx :Oocyte Number=11 Omentum :Oocyte Number=5 Sheet ovarian tissue :Oocyte Number=8 Pieces ovarian tissue :Oocyte Number=8
35 Ovarian Tissue Vitirification: New closed-type device: Cryosheet No18 Omentum :Gill s Haematoxylin stained Transplanted Ovarian tissue Oocytes Omentum
36 Ovarian Tissue Vitirification: New closed-type device: Cryosheet No21 Omentum : Transmission-electron microscopy No Ice formation Control (non-frozen) Vitrified Thawed Tissue
37 New closed-type device for ovarian tissue vitrification: Cryosheet
38 Human ovarian tissue cryopreservation: Sf vs Vit Donor case1:39years African American/ Hart Attack Brooklyn Hospital Center Donor case2:23years White/ Unknown Brookhaven Memorial Hospital Donor case3:31years White/ intracranial bleeding NYC Health Hospitals Jacobi Sf Slow freezing Slow Freezing Solution MEMα+Sucrose+Dimethyl sulfoxide+human Serum Albumin Vitrification Vit Vitrification Solution H199 +Sucrose+Ethylene Glycol +Polyvinylpyrrolidone+Synthetic Serum Substitute
39 Human ovarian tissue cryopreservation: Sf vs Vit Sf Summary of Study Design Human Ovaries Vit Thawing Culture Freezing Embedding Sectioning & Staining Follicle Counting
40 Human ovarian tissue cryopreservation: OVf vs CVit Vit vs
41 Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primordial Follicle Density, DNA DSBs and Apoptosis γh2ax positive : DNA double-strand-breaks AC3 positive : Apoptotic follicles Positive Negative H2AX H2AX AC3 AC3 Control Slow Freezing Vitrification Control vs. SF Control vs. VF SF vs. VF Primordial follicle Density (/mm3) 134.6±25 64±17 62± γh2ax+ Primordial follicle (%) 27.3± ± ± AC3+ Primordial follicle (%) 17.5± ± ± Non-apoptotic Primordial follicle density (/mm3) 112.6± ± ±
42 Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primordial Follicle Density, DNA DSBs and Apoptosis Primordial follicle Density (/mm3) * * Non-apoptotic Primordial Density (/mm3) * γh2ax+ Primordial follicle (%) NS AC3+ Primordial follicle (%) NS NS 100 NS * : Significant NS: Non Significant
43 Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primary Follicle Density, DNA DSBs and Apoptosis γh2ax positive : DNA double-strand-breaks AC3 positive : Apoptotic follicles Positive Negative H2AX H2AX AC3 AC3 Control Slow Freezing Vitrification Control vs. SF Control vs. VF SF vs. VF Primary follicle Density (/mm3) 14.6± ±2 6.6± γh2ax+ Primary follicle (%) 11.6± ± ± AC3+ Primary follicle (%) 5.8± ±3.7 26± Non-apoptotic Primary follicle density (/mm3) 14.1±5 6.2± ±
44 Human ovarian tissue cryopreservation: Sf vs Vit Impact of Vitrification vs SF on Primary Follicle Density, DNA DSBs and Apoptosis Primary follicle Density (/mm3) NS Non-apoptotic Primary Density (/mm3) NS γh2ax+ Primary follicle (%) NS AC3+ Primary follicle (%) * NS * : Significant NS: Non Significant
45 Human ovarian tissue cryopreservation: Sf vs Vit Summary and Conclusion Impact of Vitrification vs SF on Follicle Density, DNA DSBs and Apoptosis Similar follicle density Sf Similar DNA DSB Breaks Similar apoptotic activation Vit Based on histological and immunohistochemical evaluation, Sf and Vit methods appear to be comparable.
46 Human ovarian tissue cryopreservation: Sf vs Vit A Collaborative research with New York Medical Collage and Yale University to improve the ovarian tissue cryopreservation method Human Ovaries Thawing Culture Embedding Sectioning & Staining γh2ax and AC3+ Follicle Counting Freezing Now on going Data analysis RNA-sequence (NextSeq 550 illumine) cdna amplification (Nextra XT Library prep kit) RNA extraction Reversetranscription Lazer Captured Microdicection
47 Slow freezing vs vitrification Scientific Reports, 2017 Sf vs Vit
48 Slow freezing vs vitrification Scientific Reports, % CI: 0.97 [ ] Sf Vit
49 Slow freezing vs vitrification Scientific Reports, % CI: 0.71 [ ] Vit Sf
50 Slow freezing vs vitrification Scientific Reports, % CI: 1.69 [ ] Sf Vit Sf
51 Slow freezing vs vitrification Scientific Reports, % CI: 3.44 [ ] Sf Vit
52 Slow freezing vs vitrification Scientific Reports, 2017 Vit LN 2 less primordial follicular DNA strand breaks and better preservation of stromal cells.
53 Future: Ovarian tissue vitrification Device? PrOH EG DMSO PVP Glycerol PVA Trehalose Vitrification Sucrose Solution LN 2
54 Vitrificaion of human ovarian tissue?? PROS 1. Excellent survival of ovarian stroma and blood vessels 2. A fast method 3. No particular apparatuses needed 4. Also feasible clinically Ovarian tissue cryopreservation 5. Excellent survival in tissue culture CONS 1. No live births reported so far 2. Requires good training in the beginning 3. Concentrations and osmolarities and the speed of the procedure are of extremely high importance 4.Toxic influence on the tissue is possible if less trained personnel Dr. Hovatta, World Congress on Fertility Preservation 2009 in Belgium
55 Vitrificaion of human ovarian tissue?? PROS 1. Excellent survival of ovarian stroma and blood vessels 2. A fast method 3. No particular apparatuses needed 4. Also feasible clinically Ovarian tissue cryopreservation 5. Excellent survival in tissue culture CONS 1. No live births reported so far 2. Requires good training in the beginning 3. Concentrations and osmolarities and the speed of the procedure are of extremely high importance 4.Toxic influence on the tissue is possible if less trained personnel Dr. Hovatta, World Congress on Fertility Preservation 2009 in Belgium
56 Vitrificaion of human ovarian tissue?? PROS 1. Excellent survival of ovarian stroma and blood vessels 2. A fast method 3. No particular apparatuses needed 4. Also feasible clinically Ovarian tissue cryopreservation 5. Excellent survival in tissue culture CONS 1. No live births reported so far(at that time) 2. Requires good training in the beginning 3. Concentrations and osmolarities and the speed of the procedure are of extremely high importance 4.Toxic influence on the tissue is possible if less trained personnel Dr. Hovatta, World Congress on Fertility Preservation 2009 in Belgium
57 2016- ASFP (Asian Society for Fertility Preservation): countries 2015 ESHRE
58 The 1 st Congress of ASFP; Ho Chi Minh City, Vietnam
59 The 1 st Workshop and Symposium of ASFP; Shanghai, China
60 Hands on seminar: Ovarian tissue vitrification At St. Marianna University, Japan: w/member of Seoul Natl Univ In India: Fertility Preservation Society (India) New Deli, India At St. Marianna University, Japan: w/ Dr. Kemar, Dr. Mila And Dr. Kock In Indonesia: Reproductive Medicine Society Medan, Indonesia Hands on seminar and Ovariectomy and ovarian tissue vitrification for 2 young cancer patients. Almaty, Republic of Kazakhstan At St. Marianna University, Japan: w/ Dr. Novero s team and Dr. Lee
61 Hands on seminar: Ovarian tissue vitrification In Philippines: OVARIAN TISSUE CRYOPRESERVATION POST GRADUATE COURSE AND WORKSHOP. w/ Member of Dr. Novero s team Manila, Philippines At St. Marianna University, Japan: w/ Member of Dr. Sirayapiwat and Ms. Numchaisrika ASPIRE2016, Jakarta, Indonesia PROS of ovarian tissue vitrification are a fast method and no particular apparatuses needed. It is easily handled for anyone at anywhere in anytime at 第二军医大学 Pr. Lee Ovariectomy and ovarian tissue vitrification for 2 POI patients. Shanghai, China At St. Marianna University, Japan: w/ Professor Lee s team from China
62 Take home message Based on several data, Sf and Vit methods appear to be comparable. Ovarian tissue vitrification may be a promising new fertility preservation method for young cancer patients. Excellent survival of ovarian stroma and blood vessels A fast method No particular apparatuses needed Also feasible clinically Excellent survival in tissue culture It is easily handled for anyone at anywhere in anytime!!
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